The enzyme β-glucuronidase (GUS; E.C.3.2.1.31) hydrolyzes a wide variety of glucuronides. Virtually any aglycone conjugated to D-glucuronic acid through a β-O-glycosidic linkage is a substrate for GUS. In vertebrates, glucuronides containing endogenous as well as xenobiotic compounds are generated through a major detoxification pathway and excreted in urine and bile.
Escherichia coli, the major organism resident in the large intestine of vertebrates, utilizes the glucuronides generated in the liver and other organs as an efficient carbon source. In E. coli, β-glucuronidase is encoded by the gusA gene (Novel and Novel, Mol. Gen. Genet. 120: 319–335, 1973), which is one member of an operon comprising two other protein-encoding genes: gusB encoding a permease (PER) specific for β-glucuronides, and gusC encoding an outer membrane protein (OMP) that facilitates access of glucuronides to the permease located in the inner membrane.
While β-glucuronidase activity is expressed in almost all tissues of vertebrates and their resident intestinal flora, GUS activity is absent in most other organisms. Notably, plants, many bacteria, and fungi have been reported to largely, if not completely, lack GUS activity. Thus, GUS is ideal as a reporter molecule in these organisms and has become the most widely used reporter system for plants.
In addition to use as a reporter molecule, GUS in combination with an innocuous glucuronide would be a preferred system to use for positive selection of transformed plants, especially for plants that will be consumed by humans. Because of the inefficiency of methods for transforming plant cells, only a small proportion of cells actually become transformed. Thus, it is desirable to select only those cells actually transformed. Typically, the selection methods involve transforming a cell with an antibiotic resistance gene along with the gene of interest and applying antibiotics to the cells, which kills the non-transformed cells.
Consumer resistance to antibiotic resistance genes has spurned research into alternative selection systems. Positive selection systems, wherein the transformed cells contain a gene whose gene product can utilize a compound that confers a growth advantage over the non-transformed cells. Ideally both the gene and the compound are biosafe to the environment and animals and humans.
GUS is the ideal system for positive selection for many reasons. First, biosafety assessment of GUS, including ecological and toxicological concerns, has shown GUS to be safe for both the environment and consumers (Gilissen et al. Transgenic Res 7: 157–163, 1998). Second, the gus gene is already present in several de-regulated food crops, such as papaya, beet and soybean, in the United States as well as in other countries. Third, the ease of making and isolating glucuronidated compounds allows a large choice of compounds to use for conferring growth advantage.
In positive selection systems under development, sugar compounds that plants do not normally metabolize, are being exploited in combination with xylose isomerase and mannose phosphate isomerase (U.S. Pat. Nos. 5,994,629 and 5,767,378). Unfortunately, both of these systems have disadvantages: mannose is toxic to plant cells, some plants have endogenous xylose isomerase activity, and neither of the genes have undergone biosafety testing. Moreover, a reporter gene must still be used for visualization of transformed cells, a procedure that is necessary for confirmation of transformation. In addition, the intellectual property for these two systems is held by Syngenta who so far has not granted commercial licenses on terms favorable for small companies.
The gus gene in combination with a sugar glucuronide would provide the best positive selection system. GUS can serve as both a selectable and a reporter molecule; it is biosafe; and glucuronide sugars, such as cellobiuronic acid (a disaccharide comprising glucose and glucuronic acid) are readily isolated inexpensively. The E. coli gus gene, however, does not metabolize cellobiuronic acid. Therefore, there is a need for a GUS enzyme that can cleave cellobiuronic acid.
The present invention provides gene and protein sequences of fungal β-glucuronidases and variants thereof that are secreted and cleave cellobiuronic acid, while providing other related advantages.